5-oxo-1h-pyrrolo-[2, 1-c][1, 4]-benzodiazepin-2-crylamides



United States Patent 3,361,742-0X0-IH-PYRROLO-[2,1-c]{1,4]-BENZODIAZEPIN- Z-ACRYLAMIDES Julius Berger,Passaic, Andrew E. Karr, Bloomfield, Willy Leimgrnher, Cedar Grove, ArnoJohannes Schocher, Boonton, Vladimir Stefanovic, Passaic, and BenjaminTabenkin, Upper Montclair, N.J., assignors to Hoffmann-La Roche Inc.,Nutley, NJ., a corporation of New Jersey No Drawing. Filed Dec. 7, 1964,Ser. No. 416,599 9 Claims. (Cl. 260-2393) This invention relates toantibiotics and to processes for their preparation.

It is known that the cultivation of certain thermophilic actinomycetesresults in the production of a fermentation broth having anticanceractivity (see, e.g., South African application No. 63/3,875). There havenow been discovered fermentation, isolation, and conversion procedureswhich result in compounds having high antibacterial and anticanceractivity. The thermophilic actinomycetes which can be employed in theprocess of the invention are two strains of the genus Streptomyces andthe species refuineus var. thermotolerans having NRRL numbers 3143 and3144.

The above strains have the following taxonomic description:

Substrate mycelium branched, 0.5 to 0.7g in diameter. Growth at firstwhite, progressing to medium yellow, yellow-orange, or medium tan; orwhite, changing to gray, gray-green (near grey A2 fe) (Descriptive ColorNames Dictionary, Container Corporation of America), olive or black.Aerial mycelium moderate to abundant, powdery or sometimes cottony,white, cream-yellow, or medium gray to deep gray-green (near gray 24 /2ml), depending on the medium composition. Sporophores monopodiallybranched, with numerous open extended or compact spirals, the latteroccurring infrequently; spirals usually with 1 to 4 coils, sometimes upto 8 to 10 turns. Spores oval, short to elongated, irregularly rough towarty, 0.5 to 1.2 by 1.0 to 2.3a. Mycelium and spores gram-positive, notacid-fast. Vegetative mycelium lysozyme sensitive. Soluble pigmentproduced in several media. Facultative thermophile. Habitat: donkeydung.

In the specification, the color designations in parentheses areaccording to Descriptive Color Names Dictionary, Container Corporationof America.

Growth characteristics Pr0cedure.'1'he inoculum for culture studies wasdeveloped in shaken flasks for 14-16 hours at 45 C., in the medium 0.5%Bacto-Tryptone plus 0.3% Bacto yeast extract to volume with distilledwater.

The instant strains were grown at 45 C. for about 2 days, unlessotherwise stated, before their culture characteristics were determined.Cultures were then reincubated for an additional period of 3 to 10 daysto observe if any further changes occurred.

The following media designations are taken from Applied Microbiology 9:394-399 (1961).

Maintenance medium Ia NRRL 3143, growth thin, white to gray. Aerialmycelium moderate, white to light gray.

NRRL 3144, growth good, white to deep gray. Aerial mycelium abundant,white to cream-yellow to light gray.

Medium Ib NRRL 3143, growth abundant, white to light gray, later deepgray-black. Aerial mycelium moderate, thin, white to medium gray-green.

NRRL 3144, growth abundant, white to medium gray, changing to deep gray.Aerial mycelium abundant, cottony, white, later gray-green.

Medium 11 NRRL 3143, growth good, pale gray changing to deep gray.Aerial mycelium abundant, light gray to medium gray.

NRRL 3144, growth good, medium to deep gray. Aerial mycelium light gray,changing to medium gray, then greenish-gray.

Medium III NRRL 3143, growth moderate, restricted, white to pale gray.Aerial mycelium scant, appearing late, white to light gray.

NRRL 3144, growth good, white to pale cream-yellow. Aerial myceliumscant, white to cream-yellow, later light gray.

Medium IV NRRL 3143, growth thin, white to light gray. Aerial myceliumlight gray to medium gray.

NRRL 3144, growth thin, white to cream-yellow, later light gray. Aerialmycelium cream-yellow to light gray.

Medium V NRRL 3143, growth good, white then gray to light brown. Aerialmycelium thin, white at first, later pale gray-green. Soluble pigmentlight brown.

NRRL 3144, growth good, white, later cream-yellow to light brown. Aerialmycelium thin, white to creamyellow, changing to light gray-green.Soluble pigment light brown.

Medium VI NRRL 3143, growth good, relatively thick, light gray, laterchanging to dark gray-green. Aerial mycelium abundant, cottony, lightgray, becoming light gray-green. Soluble pigment yellow, later tan,light yellow reverse.

NRRL 3144, growth abundant, thick, medium gray to gray-green (near gray24 /2 fe). Aerial mycelium medium gray, later becoming gray-green (neargray 24 /2 ml.). Soluble pigment yellow, later tan, light yellowreverse.

Medium VII NRRL 3143, growth very thin, creeping, light gray. Aerialmycelium scant, white to light gray.

NRRL 3144, growth thin, creeping, light gray. Aerial mycelium scant,light gray.

Tomato oatmeal No growth with either culture at 28 C. or at 45 C.

Amidex NRRL 3144, 45 C., growth abundant, white to lemonyellow, laterdark olive. Aerial mycelium white to cream, becoming light gray. Solublepigment light tan.

NRRL 3144, 28 C., growth abundant, White to pale yellow, changing tomustard tan. Aerial mycelium moderate, light gray. No soluble pigment.

NRRL 3143, 45 C., growth abundant, white to light gray, changing to darkolive. Aerial mycelium white to light gray. Soluble pigment light tan.

NRRL 3143, 28 C., growth moderate, white, later camel (3 ie). Aerialmycelium scant, white. No soluble pigment.

NRRL 3144, 45 C., growth scant, white to pale yellow. Aerial myceliumsparse, white. No soluble pigment.

NRRL 3144, 28 C., growth fair, white to ivory, then medium olive; Aerialmycelium scant, white. No soluble pigment.

NRRL 3143, 45 C., growth very poor, white. Aerial mycelium scant, white.No soluble pigment.

NRRL 3143, 28 C., growth fair, white to pale yellow. Aerial myceliumscant, white. No soluble pigment.

Yeast and malt extract NRRL 3144, 45 C., excellent growth, white to paleyellow. Aerial mycelium white to cream. Soluble pigment pale yellow.

4. NRRL 3143, 45 C., growth scant, white to cream. Aerial myceliumtrace, white. No soluble pigment.

NRRL 3143, 28 C., growth poor, white to creamyellow. Aerial myceliumscant, white. No soluble pigment.

NRRL 3144, 28 C., abundant growth, white to pale Starch gi gg; fi gz ggggggzgi mycelium White to medi' NRRL 3144, 45 C., growth excellent, whiteto cream- O yellow, later light olive. Aerial mycelium white to cream,

NRRL 3143 45 growth abun.dant Whlte to pale then light gray. Hydrolysisgood. Trace of yellow, soluble orange-yellow. Aerial mycelium white,mealy. Soluble pigment Pigment light yellow NRRI: 3144 28 C growthexcellent white to cream- NRRL 28 Q" growth fi 3 to yellow, later lightolive. Aerial mycelium abundant, white, W Aenal mycelium modfirate W 0so u 6 later medium gray. Hydrolysis good. Trace of light yelplgment'low, soluble pigment.

Glucose nument 15 NRRL 3143, 45 C., growth moderate, white to pale NRRL3144, 45 C., growth scant, white to creamyellow. Aerial mycelium white,powdery. Hydrolysis good. yellow. Aerial mycelium trace, white. Solublepigment Trace of yellow, soluble pigment. not observed. NRRL 3143, 28C., growth moderate, white to cream- NRRL 3144, 28 C., growth moderate,white to pale yellow to tan. Aerial mycelium abundant, white. Hyyellow.Aerial mycelium scant, white. No soluble pigdrolysis good. Trace of paleyellow, soluble pigment. ment- Skim milk NRRL 3143, 45$ C., growthscant, white to pale yellow. Aerial mycelium trace, white. No solublepigment. N 3144, w growth scant, White l P Y NRRL 3143, 28 C., growthgood, white to pale yellow- 10W. Aerial mycelium scant, white.Hydrolysis good. Light brown. Aerial mycelium scant, white. No solublepigment. bfOWIl $0111b1 P Gluwse-nutriem+glycerol NRRL. 3144, 28 C.,growth moderate, white to pale yellow. Aerial mycelium, thin, white.Hydrolysis good.

NRRL 3144, 45 C., excellent growth, white to pale Light tan, solublepigment. orange-yellow. Aerial mycelium sparse, white. Soluble NRRL3143, 45 C., growth scant, white to pale pigment pale orange-yellow. 30yellow. Aerial mycelium sparse, white. Hydrolysis good.

NRRL 3144, 45 C., excellent growth, white to pale Pale brown, solublepigment. orange-yellow. Aerial mycelium sparse, white. Soluble NRRL3143, 28 C., growth fair, white to light yellow. pigment paleorange-yellow. Aerial mycelium sparse, white. Hydrolysis good. TraceNRRL 3144, 28 C., growth moderate, white, later pale brown, solublepigment.

3:522:31 tyellow. Aerial mycelium scant, white. No soluble Bennetfs agarNRRL 3143, C., growth abundant, white to pale NRRL 3144, 45 C., growthexcellent, white to pale yellow-orange. Aerial mycelium scant, white.Soluble Y g later medium tall. Aerial mycelium sandy pigment paleorange-yellow. to light gray. Trace of yellow, soluble pigment.

NRRL 3143, 28 C., growth moderate, white to golden 4.0 NRRL 3144, 3 C.,grOWth excellent, White to pale yellow, then tan. Aerial mycelium scant,white. No soluy Atifial mycelium White- No Soluble P bl pigment NRRL3143, 45 C., growth moderate, white to pale P and G dextrin yellow,later medium tan. Aerial mycelium scant, white. 7 n Trace of solublepale yellow pigment.

NRRL 3144, 45 C., growth scant, white to ale yellow. 0 1 Aerial myceliumpowdery, white. No soluble gigment. 45 NRRL 3143 28 growth ejfcenentWhlte at F NRRL 3144, 0 C growth scant, White to Pale yeL latercream-yellow. Aerial mycelium abundant, white.

low, later medium olive. Aerial mycelium moderate, N0 Soluble P white.Trace of pale yellow soluble pigment. Carrot l d potato plug NRRL 3143,45 C., growth moderate, white to cream.

Aerial mycelium white powdery No Soluble Pigment No growth was observedwith either strain at 28 C.

NRRL 3143, 28 0., growth scant, white to light yelof 45 low. Aerialmycelium poor, white. Trace of pale yellow Metabolic termites SolublePigment Hydrogen sulfide test (Bacto peptone iron agar). No

Glucose asparagine H S was produced by either strain at 28 C. or at 45C.

NRRL 3144 45 C, growth Very sparse, whim Aerial Growth of NRRL 3144 andNRRL 3143 was relatively mycelium scant, white. No soluble pigment. P onthe p p agar- NRRL 3144 Produced NRRL 3144, 28 C., growth fair, white topale cream- Yellow to Olive g aerial mycelium scant, White to yellow.Aerial mycelium moderate, powdery, White. No gray. NRRL 3143 Produced PYellow growth but no soluble pigment. aerial mycelium.

TYROSINE TEST Test Day of Aerial 1 Culture Temper- Reading GrowthMycelinm Color of Reverse Color Pigment ature, C

NRRL 3144.... 28

28 Ivory Pale salmon. 28 Pale yellow-.- Light beige. 45 Light yellowPale salmon. 45 Yellow Light vinaceous. 45 Light olive yellow Light teabrown.

NRRL3143- 2s 28 Pale salmon. 28 Light beige. 45 Pale flesh. 45 Palesalmon. 45 Fawn.

ACTION ON MILK Culture Tempera- Day of Coagulation Peptonization GrowthPigment ture, 0. Reading NRRL 3144-. 28 2 0 O 0. 28 7 0 0 Slight, lightyellow 0. 28 14 0 do 0. 28 21 45 1 0 0. 45 2 0 0. 45 4 Light beige. 45 5Visible on ring .1 D0.

NRRL 3143--.. 28 2 0 O 0 0. 28 7 0 0 Trace, light yellow 0. 28 21 45 1 00 0. 45 2 0 0 0. 45 4 0 Few ring colonies, Light beige.

(incomplete) light yellow. 45 5 do Do.

Proteolytic activity No liquefaction of gelatin was observed at 28 C. orat C. with either culture. All inoculated gelatin tubes were incubatedfor 10 days at 45 C. or 14 days at 28 25 C. But high proteolyticactivity vs. many proteins was observed on complex media.

Nitrate reductase activity Organic br0th.NRRL 3144, 45 C., surface andsubmerged growth recorded in 48 hours; no nitrate reduction observedafter 6 days.

NRRL 3144, 28 C., surface and submerged growth observed in 7 days; nonitrate reduction after 14 days.

NRRL 3143, 45 C., surface and submerged growth developed in 48 hours; nonitrate reduction observed in 6 days.

NRRL 3143, 28 C., submerged growth observed in 7 days; no nitratereduction after 14 days.

Inorganic br0th.NRRL 3144, 45 C., light submerged growth observed in 4days; no nitrate reduction after 6 days.

NRRL 3144, 28 C., surface and submerged growth recorded in 7 days; nonitrate reduction after 14 days.

NRRL 3143, 45 C., no visible growth detected during 6 day period.

NRRL 3143, 28 C., some submerged growth recorded in 7 days; no nitratereduction after 14 days.

NITROGEN UTILIZATION TEST Growth Observations Test Compound NBRL 3144NRRL 3143 28 C. 45" C. 28 C. 45 C ii-+ 6O Betaine HCI a-Aminoisobutyricacid :1: :1:

Creatine i iit i- 7 O Explanation of signs:

growth like control, i.e., with glucose but without nitrogen source. :1:poor growth. poor to fair growth. fair growth. good growth Incubationtime: 45 C.65 hours; 28 C.100 hours.

CARBON UTILIZATION TEST Growth Observations Test Compound NRRL 3144 NRRL3143 28 C. 45 C. 28 C. 45 C.

Rhamnose- Dextrose- Fructose Saccharoso Lactosei! l l [+++-FCellobiose-- Dextrin. Starch- Na-sucoinate Explanation of signs:

growth like control, i.e., with (NHOZSO but without carbon is poorgrowth.

(+) poor to fair growth.

+ fair growth.

+-|- good growth.

Incubation time: 45 C.65 hours; 28' C. hours.

The fermentation process of the invention is carried out by culturingone of the above organisms in a nutrient medium. The characteristics ofthe nutrient media are highly important for obtaining good yields of theantibiotic of the invention. In particular, the nutrient media must havethe following characteristics in order to give good antibiotic yields:

(1) From about 0.1 to about 9.0, preferably from about 1.0 to about 5.0percent by weight of complex protein-containing products, in the finalnutrient medium. The protein source should have a ratio of totalnitrogen to amino nitrogen greater than 3 and the total nitrogen in themedium should be in the range of from about 0.005 to about 0.48,preferably from about 0.015 to about 0.20 percent W./v. Examples ofprotein sources that answer the above requirements include milkproducts, e.1g., pep tonized milk or dried milk, skim milk, etc.;enzymatic digests of soy, yeast, fish or peanut proteins, or mixturesthereof.

(2) A carbohydrate source. The quantity of carbohydrate does not appearto be too significant and is generally in the range of from about 0.25to about 11.5 percent w./v. Carbohydrate sources that can be employed 7include alcohols, sugars and crude or purified starches, e.g., cornstarch, potato starch, soluble starches, glycerol, mannitol, lactose,maltose, etc.

(3) The initial pH of the fermentation broth must be in the range offrom about 6 to about 8.5, preferably from about 6.7 to about 7.3. Afterabout 12 to about 15 hours of fermentation, the pH is maintainedthereafter in the range of about 6.0 to about 6.9, e.g., by the additionof an alkali metal hydroxide, e.g., sodium hydroxide, or by sulfuricacid.

Also, there can be added up to about 2.5 percent of dried yeast or yeastextract to the basal medium. However, this is an optional ingredient.

The fermentation is carried out by forming a nutrient medium asdescribed above, sterilizing it, e.g., at 120 C. for 15 to 20 minutes,and then inoculating the nutrient medium either with spores orvegetative growth of. one of the above organisms and permitting thefermentation to proceed with agitation and aeration at a temperature inthe range of from about 35 to about 55 preferably from about 40 to about50 C. and from about to about 100, preferably about 12 to about 30hours.

The fermentation broth is then optionally filtered, and extracted withan extraction solvent, preferably in two extraction steps.

Extraction solvents that can be employed in both extraction stepsinclude (a) a solvent comprising a monohydric saturated aliphaticalcohol having a boiling point up to about 200 C. which has a lowmiscibility (e.g., less than 25 percent) with cold water, e.g.,n-butanol,- isobutanol, n-pentanol, n-hexanol, 4-methyl-2-1pentan01,etc. This solvent can be entirely one of said alcohols or a mixture ofsaid alcohols or can be a homogeneous mixture of one or more of saidalcohols with up to about 90 percent by volume of another solvent havinga low miscibility with cold water such as a monoor polyhalogenatedhydrocarbon having a boiling point between about 40 and about 200 C.,e.g., chloroform, carbon tetrachloride, methylene chloride, ethylenedichloride, etc. The halogen substituents can be chloro, bromo, iodo, orfluoro. Other solvents that can be employed in mixtures with alcoholsinclude ethers, esters, ketones, aromatic hydrocarbons, etc., which havea low miscibility with cold water.

In the first extraction step, the solvent to fermentation broth ratioemployed is in the range of from about 4:1 to about 1:10, depending onthe equipment used, the solvent employed, and the degree of extractiondesired. The ratio for batch extraction is usually about 1:1 to about4:1. The ratio for column counter-current extraction is usually about1:2 to about 1.3:1. A pH in the range of from about 4 to about 10,preferably about 6,

phase is introduced near the top of the column, and a solvent phaseoptionally in equilibrium withwater is introduced near thebottom of thecolumn. The solvent to water ratio is in the range of from about 1:5 toabout 1:15,

preferably about 1:10. The feed solution to water ratio is in the range,of about 1:30. Most of the product is ex.- tracted into the solventphase which leaves the topof' the column.

n-Butanol'is' the preferred solvent for use in both of the aboveextraction steps. When n-butanol is employed,

about /z to about 1 percent of the total solids dissolved in the.fermentation brothpass into the n-butanol'phase.

This percentage of the solids contains most of the product.

The above steps of extracting the broth with a solvent, concentratingthe solvent extract, and carrying out a fractional liquid extraction ofthe concentrate can also be carried out by omitting the concentratingstep, although this process is much less efiicient. Also, the secondextraction step above can be omitted, but the yield of product may bedecreased somewhat.

The above solvent extract obtained from the fractional liquid extractioncolumn is concentrated by evaporation to a solution or paste containingabout 5 to about 20 percent solids; then about 25 to about 50 volumes ofnhexane are added, and the resulting slurry filtered. The precipitatedproduct is then vacuum-dried to give a solid compound in the form of apartially purified yellow powder of about 45 to about 75 percent purity.This compound is useful in the same manner as the compounds of FormulasI, II, 1H, and IV disclosed hereinafter, with dosages adjusted to animalresponse.

The compound of from about 45 to about 75 percent purity, obtainedabove, is further purified by one of the following techniques:

(a) The 4575 percent pure compound is partitioned between a solventsystem comprising either a chloroformlower alkanol-water system or anethyl acetate-water system. Examples of solvent systems useful in thepractice of the invention include chloroform:isopropanolzwater (1:122v./v. mixture), ethylacetatezwater (19.5:9),chloroform:isopropanolzethanol:water (3:1: 1:5 v./v.chloroform:isopropanol:methanolzwater (3:1:lz5 v./v), etc. Thepartitioning is carried out in e.g., a Craig countercurrent distributionapparatus, e.g., having 200 tubes, or in an extraction column. When thedistribution is carried out in the Craig apparatus, the compound isdissolved in one of the above solvent systems, and placed in one or moreof the first tubes of the Craig apparatus. The solutions in those tubesshowing a UV. maximum at 330-335 my and significant in vitro activityagainst Bacillus sp. TA (NRRL 13-3167) are combined, and the phasesseparated. In place of Bacillus sp. TA, various other organisms canequally well be employed, for example, Staphylococcus aureus, Sarcinalutca, Bacillus subtilis, Escherichia coli bacteriophage T-5, etc. Theorganic solvent phase is evaporated to dryness at a tem perature belowabout 35 C. and the aqueous phase evaporated to a small volume, also ata temperature below about 35 C. The residues from both phases arecombined and lyophilized, affording a compound which is about percentpure.

This material is obtained in purified amorphous form by dissolving theabove 90 percent pure compound in a nonalcoholic organic solvent, e.g.,acetonitrile, dimethylfOrmamide, dimethylsulfoxide, C -C ketones, suchas acetone, etc., and then fractionally precipitating the product by theaddition of an aliphatic hydrocarbon, e.g., petroleum ether, hexane,etc.

The 80-90 percent pure compound and the purified amorphous compound arealso useful in the same manner as the compounds of. Formulas I, H, III,and IV disclosed hereinafter.

(b) The 45-75 percent pure compound is dissolved in methanol in amountto form from about 2 to about 20 percent solution. The solution isfiltered, then about 0.05 to about 0.5, preferably about 0.4, volumes ofwater per volume of methanol are added. The solution'is then cooled to atemperature ranging from about -8 C. to about 20 C. Theresulting. slurryis filtered, optionally dried, and then dissolved in methanol toformfrom about 1 to about 20 percent, e.g., about 2 percent, solution. Thesolution is filtered and then from about 0.01. to about 0.3, preferablyabout 0.15, volumes of'water per volume of methanol are added slowlywith cooling, e.g., at a tem- 1 Northern Utilization Research andDevelopment Division, U.S. Department of Agriculture, Peoria, 111.

OH cm cone:

5,10,11,1 1a tetrahydro-9-hydroxy-1 1-methoxy-8 methyloxo1H-pyrrolo-[2.1-c] [1,4]-benzodiazepin-2-acrylamide. X is a number fromO to 1.5. The above step can also be carried out successfully with aneven less pure product, e.g., with a product of only about 20% purity.

The compound of Formula I or the purified amorphous material obtained byprocess (a) above can be heated in a nonalcoholic inert solvent, e.g.,dimethylsulfoxide, acetonitrile, acetone, etc., boiling from about 50 C.at a temperature in the range of from about 50 to about 200 to form acompound of the formula H The compound of Formula II can then be reactedwith either (a) water at a temperature in the range of from about 0 toabout 100 C. or (b) with an alcohol having at least one OH group, e.g.,an alcohol of the formula ROH wherein R is lower alkyl, phenyl loweralkyl, or amino lower alkyl, e.g., methyl, ethyl, butyl, benzyl,fl-aminoethyl, etc.; or a dior polyhydric alcohol, e.g., a sugar,mannitol, ethylene glycol, etc.

When water is employed, the product is a crystalline compound of theformula III When alcohol is used as the reactant, the product obtainedhas the formula wherein R is lower alkyl, phenyl lower alkyl, aminolower alkyl, or a dihydric or polyhydric alcohol residue.

It is to be noted that the compounds of Formulas I and IV aresignificantly more stable than the compound of Formula III and hence thecompounds of Formulas I and IV are highly preferred for use in thepractice of the invention.

The compounds of Formulas I, II, In, and IV are seful as anticanceragents, e.g., they are active against S180 and Ehrlich solid tumors inmice, antiprotozoal agents, e.g., against Endamoeba histolytica and Trichomonas vaginalis, and anthelmintic agents, e.g., against Syphaciaobvelata. They can be administed orally or by injection, e.g., in theform of an aqueous solution intravenously, such as by slow intravenousdrip of a 0.01 percent aqueous solution. Typical intravenous dosagesrange from about 0.01 to 0.05 mg./kg. animal body weight, and typicaloral doses are in the range of about 0.22.0 mg./kg. animal body weight,with the dosage adjusted to species and individual requirements.

The process of the invention is illustrated by the following exampleswhich are not meant to limit the invention.

EXAMPLE 1 Several loopfuls of spores of Streptomyces sp. NRRL 3143 aretransferred from a mature (2-3 day old) 45 stock agar slant to ml. ofgermination medium composed as follows in gm. per liter:

Bacto tryptone gm 5 Bacto yeast extract gm 2 Bacto soytone gm 2 Solublestarch gm 10 Mannitol gm 5 Magnesium su1fate-7H O mg 200 Ferrousammonium sulfate-6H O mg 10 Zinc chloride mg 2.1 Manganous chloride'4I-IO mg 1.8 Copper sulfate-5H O mg 0.3 Cobalt nitrate-6H O mg 0.5 Boric.acid mg 0.6

The medium is contained in a 1000 ml. Pyrex Blake bottle. Afterinoculation the medium is incubated at 45 C., with constant vigorousagitation, on a rotary shaker for 16 hours.

During this period a vigorous growth of the organism ensues.

The contents of two such incubated Blake bottles are pooled into a 500ml. Pyrex inoculum transfer bottle fitted with a tubulature at thebottom and containing ml. of sterile water.

The entire contents of the inoculum transfer bottle are transferred to a100-gallon stainless steel fermentor which is prepared for it asfollows:

To 25 gallons of clean tap water contained in a stainless steelfennentor fitted for controlled agitation, aeration, and temperaturecontrol are added the following in egredients Shefiield NZ-Amine B.Difco Bacto Soytone.

* Difco Bacto yeast extract.

When all the ingredients are dissolved the volume is brought to 40gallons (150 liters) with tap water and the speed of 400 rpm, the Whilemaintaining a tempera- 10 ture of 48 C.

12 EXAMPLE 2 A series of fermentors are prepared With the same basicmedium and inoculated as described in Example 1 except that variouscommercial sources of enzyme-hydrolyzed soy protein and various sourcesof whole dried yeast,

yeast extract, or yeast autolyzate are used in the medium wherehydrolyzed soy protein and aqueous extract of yeast were indicated. Allmedia but one give good broth protencies but some media are noticeablysuperior, as may be seen from the following table:

HARVEST POTENCIES REACHED IN MEDIA CONTAINING VARIOUS SOURCES OFHYDROLYZED SOY PROTEIN AND YEAST ADDITIVES Maximum Broth Potency Reachedin units/ml.

Yeast Additive Shefiield Soy T Hydrolyzate Amber Hydrolyzed Soy ProteinS. Sarcina Bacillus S. Sarcimz Bacillus aureus Zutea sp. TA aureus Zuteasp. TA

Amber BYF-SO 980 930 1, 200 710 720 820 Amber BYE-300 930 760 1, 400 l,200 l, 200 1, 900 National Grain Yeast Autlyzate 870 930 l, 400 860 9901, 400 Basamin Busch 1, 100 970 770 1, 100 1, 400 1, 200 Standard BrandsDried Brewers Yeast 850 890 1, 300 980 920 1,200 Anheuser Busch DriedDebittered Yeast 700 630 960 l, 100 1,100 1, 900 Anheuser Busch PrimaryDried Yeast Did not support growth 690 710 1, 000

Foam is controlled by the addition, as needed, of a sterile 2.5%suspension of Dow Silicone Emulsion AF. About 3000 ml. of defoamersuSpenSiOn is used during the batch. Hourly samples are takenaseptically from the 12th hour on and assayed for in vitro potency. Thedata obtained on the above batch is shown in the table which follows.This batch reaches its maximum potency in 18-20 hours.

The product is isolated from the above fermentation media according tothe process of Example 1.

EXAMPLE 3 Twenty-five gallons of clean tap water are placed into Theabove process is repeated ten times, the broths obtained are combined,the pH is adjusted to 6, the combined broths are filtered, and theresulting filtrate is extracted countercurrently at the rate of 128gallons per hour with about the same rate of butanol, in a 12" diameterby 11 ft. high Karr extraction column. A water backwash of 0.2 times thebutanol rate is employed at the top of the extraction column to minimizethe carry-over of Water soluble components. The butanol extract isconcentrated to approximately a 5 percent solution which comprises thefeed to the center of a 3 diameter by 20 ft. high Karr fractional liquidextraction column. This column is operated at a water to butanol ratioof about to 1, and the butanol extract contains the product. The butanolextract is concentrated by evaporation to a solution or paste containingabout 5 to about percent solids; then about to about volumes of n-hexaneare added, and the resulting slurry filtered. The precipitated productis then vacuum-dried to give a solid compound.

a -gallon fermentor and used to dissolve the following ingredients:

The volume is brought to 40 gallons with tap water and the pH adjustedto 7.2 with 5 N potassium hydroxide.

The fermentor is then closed and sterilized by heating to C.'for 60minutes. The batch is cooled to 48 C. and inoculated as described inExample 1. The air flow 1s adjusted to 3 c.f.m., shaft speed 400 rpm,and the temperature maintained-at 48 C. throughout.

The course of the fermentation may be seen from the data in the tablewhich follows.

Age in Hours Bacillus sp. TA, pH

units per ml.

The product is isolated from the above fermentation broth as describedin Example 1.

EXAMPLE 4 Three identical 100-gallon fermeutors are each charged with 40gallons of nutrient medium, the composition of which is as follows:

- Percent Hydrolyzed soy protein (Amber HSP 75) 0.2 Water-solubleportion of autolyzed yeast (Amber BYF 300) 0.2 Tryptic digest of casein(NZ Amine B) 0.5 Potato starch 1.0

Magnesium sulfate heptahydrate 0.02 Trace elements-as described inExample 1.

All batches are adjusted to pH 7.0 before sterilization.

The tanks are sterilized at 120 C. for one hour, cooled to 48 C. andtanks 2 and 3 are each inoculated with 150 ml. of 18 /2 hour growthtaken from a batch of culture NRRL 3143 developed on the same mediumcomposition and operating conditions as described for Example 1.

After inoculation, the tanks are each aerated at the rate of 3 cubicfeet per minute, agitated at a shaft speed of 400 r.p.m., and maintainedat a temperature of 48 C.

At the 12th hour of incubation Tank 2 is adjusted to a pH of 6.8 andacid or alkali is added periodically to maintain the pH between pH 6.7and 6.9.

Also at the 12th hour, Tank 3 is adjusted to pH 7.2 and the pH ismaintained between 7.2 and 7.4 for the duration of the fermentation bythe addition, as needed, of appropriate amounts of acid or alkali.

The pH of Tank 1 is allowed to go unadjusted.

It can be seen from the data in the following table that pH control bothduring the fermentation and thereafter is important.

14 EXAMPLE 5 7 g. of the product prepared by the process of Example 1 isdissolved in 350 ml. of chloroform-isopropanol (1:1) which waspreviously equilibrated with water. This solution is introduced into thecenter of a one-inch diameter by twenty-foot high Karr extraction columnat the rate of 0.5 mL/min. Simultaneously mL/min. of equilibratedaqueous phase is introduced at the bottom of the column and 14.6ml./min. of equilibrated solvent phase is introduced at the top of thecolumn. Most of the product is extracted into the aqueous phase leavingthe top of the column. The aqueous phase is re-extractedcountercurrently in a one-inch diameter by ten-foot high extractioncolumn employing a 1:1 isopropanol-chloroform solvent at a solvent towater ratio of 1:1. The product passes into the isopropanol-chloroformsolvent which leaves at the bottom of the extraction column. Thissolution is concentrated to 58 ml., filtered, and then treated with 2.9liters of technical hexane to precipitate the product. The product isfiltered and dried.

The dried product which is percent pure is crystallized from acetone toyield a highly active purified material.

EXAMPLE 6 20 g. of the product prepared according to Example 1 wasdistributed between ml. each of upper and lower phases of a solventsystem consisting of isopropanol; chloroform2water (121:2). The phaseswere then introduced into the first three tubes of a ZOO-tube Craigcountercurrent distribution apparatus and run at room temperature for197 transfers. The upper and lower phases of tubes 45 to 75 werecombined, the organic phase evaporated at a temperature below 35 C. invacuo and the residue added to the corresponding aqueous phase, and theaqueous phase lyophylized. The lyophylized residue was crystallized fromacetone to yield a purified material.

EXAMPLE 7 200 g. of the product prepared according to Example 1 isdissolved in 4000 ml. of methanol and the solution filtered. Thefiltrate is agitated and 332 ml. of water added during the course of onehour. The temperature during the addition is maintained at 22 C. Thesolution is then cooled to 0 C. in one hour and then stirred overnightin a cooled bath maintained at -9 C. Then the resulting slurry isfiltered, washed twice with a minimum quantity of aqueous methanol (8.3volume/volume percent H 0), and dried. in a vacuum dessicator at roomtemperature. 86.9 g. of dark brown precipitate is obtained.

The above precipitate is dissolved in 4340 ml. of methanol, filtered,and 672.7 m1. of water slowly added over a period of 3 hours at atemperature of 0 C. The resulting mixture is then placed in a cooledbath at INFLUENCE 0]? pH CONTROL OF THE BATCH ON THE YIELD AND STABILITYOF THE ANTIBIOTIC Broth Potency in units (TA) per ml.

Tank

No. Treatment Age in Hours 1 No pH adjustment 370 580 780 1, 100 1, 000730 700 630 400 pH of broth 6. 9 6. 9 6. 9 6. 8 6. 8 7. 1 7. 1 7. 0 7. 3

2 pH maintained at 6.76.9 from 0 290 700 1, 400 2, 600 3, 000 3, 000 2,900 3, 000 2, 900 3, 200 2, 900 2, 7 00 12th hour on.

pH of broth 6.8 6. 8 6.8 6. 8 6. 8 6. 8 6. 8 6. 9 6. 7 6. 7 6.7 6, 7 6.9

3 p11 maintained at 7.2-7.4 from O 420 910 1, 400 4, 200 4, 600 4, 3003, 000 2,700 1, 800 1, 600 1. 600

12th hour on.

pH of broth 7. 2 7. 2 7. 2 7. 2 7. 2 7. 2 7. 2 7. 3 7. 3 7. 2 7. 2 7. 37. 4

15- 9 C. and then stirred overnight. The crystals are then filtered otf,washed with a minimum of aqueous methanol at .20 C. (15.5 volume/volumepercent H and vacuum dried at room temperature. 69.8 g. of a yellowneedlelike crystalline solid is obtained. The crystalline solid is againdissolved in 4340 ml. of methanol, 6.95 g. activated charcoal (Norite56) are added with stirring and after minutes at room temperaturefiltered off and the filtrate treated with 672.7 1111. of water addedslowly over a period of 3 hours at 0 C. The mixture is then placed in acooled bath at 9" C. and stirred overnight. The resulting crystals arefiltered, washed with a minimum of aqueous methanol at C. and vacuumdried at room temperature. 59.6 g. of a light yellow crystalline productof Formula I is obtained:

The product of Formula I was recrystallized from acetonitrile to preparethe corresponding anhydrous compound of the formula EXAMPLE s EXAMPLE 90.1 g. of the compound of Formula 11 obtained in the above Example 8 isdissolved in 50 ml. of boiling acetonitrile. An excess of Water wasadded and the mixture allowed to stand for one hour at room temperature.The resulting solution is concentrated by distillation to a small volumeat room temperature under vacuum. On standing, crystals of the compoundof Formula III are obtained which melt at 188-194 dec. (vac.):

III

1 6 EXAMPLE 10 mg. of the compound of Formula II obtained in Ex- 1 ample8 above is dissolved in 15 ml. of n-butanol at room temperature. Theresulting clear solution is concentrated to a small volume at roomtemperature under high vacuum. The residue is treated with 30 ml. ofnhexane. A product having the formula precipitated out, melting point155-175 C. with decomposition (vac.)

EXAMPLE 11 0.2 g. of the compound of Formula II obtained in Example 8above is dissolved in ml. of boiling acetonitrile and 5 ml. of benzylalcohol added thereto. The resulting solution is allowed to stand for'3' hours at room temperature. The acetonitrile is removed under vacuumat room temperature. The residue is treated with 100 ml, of ether. Aproduct having the formula VI OCH2 0 CONHg com I wherein R is selectedfrom the group consisting of lower alkyl, phenyl lower alkyl, and aminolower alkyl.

2. A compound having the formula:

0H OCH;

3. A compound having the formula:

on H ecu,

CONE;

17 18 4. A compound having the formula: 8. A crystalline form of thecompound of claim 7. (682,363: 9. A proces for the preparation of acompound of the formula:

5. A compound having the formula:

53 wherein R is selected from the group consisting of lower alkyl,phenyl lower alkyl, and amino-lower alkyl,

comprising reacting a compound of the formula:

CONE:

with an alcohol selected from the group consisting of a lower alkanol,phenyl lower alkanol, and amino-lower alkanol.

References Cited UNITED STATES PATENTS 3,247,187 4/1966 Fryer et al260-239.3

WALTER A. MODANCE, Primary Examiner.

R. T. BOND, Assistant Examiner.

1. A COMPOUND HAVING THE FORMULA: